Pteridine salvage throughout the Leishmania infectious cycle

Molecular & Biochemical Parasitology 113 (2001) 199–213

Pteridine salvage throughout the Leishmania infectious cycle:implications for antifolate chemotherapy�

Mark L. Cunningham, Stephen M. Beverley *Department of Molecular Microbiology, Washington Uni6ersity School of Medicine, St Louis, MO 63110, USA

Received 10 November 2000; received in revised form 3 January 2001; accepted 5 January 2001

Abstract

Protozoan parasites of the trypanosomatid genus Leishmania are pteridine auxotrophs, and have evolved an elaborate andversatile pteridine salvage network capable of accumulating and reducing pteridines. This includes biopterin and folatetransporters (BT1 and FT1), pteridine reductase (PTR1), and dihydrofolate reductase–thymidylate synthase (DHFR-TS).Notably, PTR1 is a novel alternative pteridine reductase whose activity is resistant to inhibition by standard antifolates. Incultured promastigote parasites, PTR1 can function as a metabolic by-pass under conditions of DHFR inhibition and thus reducethe efficacy of chemotherapy. To test whether pteridine salvage occurred in the infectious stage of the parasite, we examinedseveral pathogenic species of Leishmania and the disease-causing amastigote stage that resides within human macrophages. Toaccomplish this we developed a new sensitive HPLC-based assay for PTR1 activity. These studies established the existence of thepteridine salvage pathway throughout the infectious cycle of Leishmania, including amastigotes. In general, activities were not wellcorrelated with RNA transcript levels, suggesting the occurrence of at least two different modes of post-transcriptional regulation.Thus, pteridine salvage by amastigotes may account for the clinical inefficacy of antifolates against leishmaniasis, and ultimatelyprovide insights into how this may be overcome in the future. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Biopterin transporter; Folate transporter; Developmental regulation; Dihydrofolate reductase; Pteridine reductase; HPLC biopterinassay

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1. Introduction

Trypanosomatid parasitic protozoa of the genusLeishmania are the causative agents of a broad range ofhuman diseases, ranging in severity from self-limitingcutaneous lesions to fatal visceral infections. An esti-mated 15 million people are infected with a further 350million at risk in tropical and sub-tropical regions ofthe world [1]. Leishmania are vector-borne parasitestransmitted by phlebotomine sand flies. Within the fly,

they reside within the alimentary tract where they repli-cate. Upon entry into stationary phase a proportion ofthe cell population differentiates from the non-infec-tious promastigote stage into the highly infectiousmetacyclic form. After a sand fly bite, metacyclics aredeposited into the mammalian host where they aretaken up by macrophages. Here, they differentiate intothe rapidly dividing, non-flagellated amastigote stagewithin an acidified phagolysosome. Existing chemother-apy is unsatisfactory, relying upon antiquated pentava-lent antimonials such as Pentostam despite considerablehost toxicity and some evidence for the emergence ofparasite resistance [2]. Moreover, safe vaccines are notavailable. Consequently, there is considerable interest inthe identification and characterization of novel bio-chemical pathways with the aim of developing newchemotherapies.

Leishmania, in contrast to their mammalian hosts,are pteridine auxotrophs and therefore have an abso-lute requirement for an exogenous source [3–7]. To

Abbre6iations: BT1, biopterin transporter 1; DHFR-TS, dihydrofo-late reductase–thymidylate synthase; FT1, folate transporter 1; H2-biopterin, dihydrobiopterin; H4-biopterin, tetrahydrobiopterin;H2-folate, dihydrofolate; H4-folate, tetrahydrofolate; PTR1, pteridinereductase 1.� Note: Nucleotide sequence data reported in this paper are avail-

able at the GenBank™ database under the accession numberAAB61214

* Corresponding author. Tel.: +1-314-7472630; fax: +1-314-7472634.

E-mail address: [email protected] (S.M. Beverley).

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -6851 (01 )00213 -4

M.L. Cunningham, S.M. Be6erley / Molecular & Biochemical Parasitology 113 (2001) 199–213200

overcome this deficiency, Leishmania has evolved acomplex and versatile pteridine salvage network capa-ble of scavenging a wide array of conjugated andunconjugated pteridines, notably folate and biopterin,respectively (see Fig. 1). Two distinct plasma membranetransporters mediate the active accumulation of pteridi-nes: folate uptake occurs predominantly via the folatetransporter (FT1) (Moore JB, Beverley SM, manuscriptin preparation), whereas biopterin transport occurs ex-clusively via biopterin transporter 1 (BT1) [8–10](Moore & Beverley, manuscript in preparation). Whilstthe active uptake of folates by transporters is wellknown, BT1 is the only known example of an unconju-gated pteridine transporter (for reviews see [11,12]).

Folate and biopterin serve as cofactors only in theirfully reduced tetrahydro-forms, H4-folate and H4-biopterin, respectively. In Leishmania and mammaliancells, H4-folate is generated from folate and H2-folateby the NADPH-dependent enzyme dihydrofolate re-ductase (DHFR) [13]. In Leishmania and other proto-zoans, DHFR occurs as a bifunctional enzyme, joinedto thymidylate synthase (DHFR-TS) [14–16]. The prin-cipal role of H4-folate is as an essential co-factor in thede novo synthesis of thymidylate in Leishmania [16];correspondingly, genetic deletion of DHFR-TS is lethalin the absence of thymidine [17] and DHFR-TS knock-out parasites are not able to establish infections in mice[18].

In mammalian cells H4-biopterin is synthesized denovo, or salvaged through DHFR-mediated reductionof H2-biopterin [19]. In contrast, in Leishmania the denovo biopterin synthetic pathway is absent [3–7] andDHFR-TS shows no activity with biopterin or H2-biopterin [20]. Instead, reduced biopterin is generatedthrough the action of the novel enzyme Pteridine Re-ductase 1 (PTR1), which sequentially reduces oxidizedbiopterin to dihydro- (H2-biopterin) and then H4-biopterin [20–23]. This NADPH-dependent enzyme isstructurally unrelated to DHFR and belongs to theshort-chain dehydrogenase family [22–26]. PTR1 ex-hibits a broad specificity for pteridine substrate and willalso reduce folate to the H2- and H4-forms [20–22].Deletion of the PTR1 gene is lethal to the insect stagepromastigotes but can be offset by provision of reducedpterins but not folates, indicating an essential role forunconjugated pteridines [20–22]. While H4-biopterin isan essential cofactor in many reactions including etherlipid cleavage, aromatic amino acid hydroxylations,molybdopterin synthesis and nitric oxide synthesis inhigher eukaryotes [27–30], the role(s) of H4-biopterin inLeishmania has not been clearly established, althoughinvolvement in oxidant resistance has been proposed(Nare et al., manuscript in preparation).

Despite reduced folate and biopterin being essentialfor growth, anti-pteridines have not shown much

Fig. 1. Pteridine salvage in Leishmania. The diagram shows the transporters responsible for pteridine accumulation, and their subsequent reductionand utilization within Leishmania metabolism. BT1, biopterin transporter 1; FT1, folate transporter 1; PTR1, pteridine reductase 1; DHFR-TS,bifunctional dihydrofolate reductase–thymidylate synthase (the activities are shown separately in the figure); SHMT; serine trans-hydroxymethyltransferase; DHPR, dihydropteridine reductase; q-H2-biopterin, quinonoid dihydrobiopterin, and ‘?’, unknown enzyme(s) utilizing H4-biopterin.The widths of the arrows indicate the relative contributions of each enzyme in steps where more than one enzyme is implicated [20]. Please seethe text for literature references.

M.L. Cunningham, S.M. Be6erley / Molecular & Biochemical Parasitology 113 (2001) 199–213 201

promise clinically against Leishmania in contrast toother protozoal infections [5,31–36]. Our current un-derstanding of pteridine metabolism is almost entirelybased on studies of the promastigote (insect) stage ofthe parasite, as this is readily cultivated in quantitiessufficient for biochemical studies. From these studies,we have proposed that enzymes of the pteridine salvagepathway may be responsible for the poor efficacy ofanti-pteridines against Leishmania promastigotes[20,21,37]. In this work, we have extended these studiesto pteridine salvage within the infectious cycle of threedifferent Leishmania species, including ones responsiblefor cutaneous (L. major, L. mexicana) and fatal visceraldisease (L. dono6ani ). We made use of the uniqueproperties of each species in culture to gain access totwo key developmental stages. With L. major, we wereable to study the infectious metacyclic stage, whicharises within the sand fly prior to transmission tovertebrates. With L. mexicana, we were able to makeuse of its ability to differentiate in vitro to an amastig-ote-like form that closely resembles amastigotes recov-ered from infected macrophages, the ultimate target ofprospective chemotherapy. These studies establish theimportance of pteridine salvage pathways throughoutthe infectious cycle of this deadly parasite and providea clearer understanding of the requirements for success-ful anti-folate based chemotherapy.

2. Materials and methods

2.1. Reagents

[3%,5%,7,9-3H]-Folate, [3%,5%,7-3H]-methotrexate and[3H(G)]-biopterin (randomly labeled) were purchasedfrom Moravek. Dihydrobiopterin (H2-biopterin) waspurchased from Schircks Laboratories. Folate-deficientmedium (fdM199) was custom-manufactured by Gibco-BRL and comprises of M199 with Hanks salts andlacking both folate and thymidine [20]. Fetal calf serumwas purchased from Bio-Whittaker, and embryonicbovine fluid was from Sigma. Trypticase was purchasedfrom Becton Dickinson. All other reagents were ofanalytical grade.

2.2. Leishmania culture

The strains of Leishmania used were: L. majorFriedlin V1 (MHOM/JL/80/Friedlin), L. major CC-1[38], a null mutant of CC-1 lacking PTR1 (ptr1−) orover-expressing PTR1 [21], L. dono6ani Sudanese strain1S2D (MHOM/SD/00/1S-2D) and L. mexicana(MNYC/BZ/62/M379). In this work, all species and/orstrains of Leishmania have been shown in previousstudies to be fully virulent. Promastigotes were main-tained by serial passage in M199 medium supplemented

10% heat-inactivated fetal calf serum at 26°C as de-scribed [38]. This medium was further supplementedwith 2 mg ml−1 H2-biopterin for growth of L. majorptr1− and geneticin (G418; 10 mg ml−1) for growth ofthe PTR1-over-expressing line. L. mexicana axenicamastigotes were grown at 34°C in 5% CO2 in asimplified version of JH-3 medium [39] developed byDavid Russell (Washington University, St Louis). Theaxenic amastigote medium consists of M199 supple-mented with 0.25% glucose, 0.5% trypticase, 0.1 mMadenine, 0.0005% hemin, 0.075% L-glutamine, 0.0001%biotin, 10% heat-inactivated fetal calf serum and 10%heat inactivated embryonic bovine fluid. Axenicamastigotes were obtained by diluting late log-phase L.mexicana promastigotes sixfold into axenic amastigotemedium, and incubating at 34o C in the presence of 5%CO2 for 5 days, by which time differentiation wascomplete. Axenic amastigotes were maintained by serialpassage every 4 days. Logarithmic, early stationary and48 h stationary phase promastigotes were taken whencells reached 3–6×106 ml−1, maximal density (typi-cally 2.5×107) and 48 h after reaching maximal den-sity, respectively. Amastigotes were analyzed in eitherlogarithmic (1–3×106 cells ml−1) or 48 h after entryinto stationary phase (typically 2.5×107cells ml−1).Metacyclic promastigotes were isolated from 48 h sta-tionary phase cultures of L. major by virtue of theirinability to react with peanut agglutinin as described[40].

2.3. Purification of radiolabeled ligands

[3H]-Folate and [3H]-methotrexate were purified byHPLC as described previously [41] on an octadecylsi-lane column using a mobile phase of 10 mM trifl-uroacetic acid with a 0–20% gradient of acetonitrile, 8mM trifluroacetic acid. [3H]-Biopterin was purified byHPLC on an octadecylsilane column using an isocraticmobile phase of 10% methanol. Purified isotopes werealiquoted and stored at −80°C for up to 6 months,without significant breakdown.

2.4. Preparation of cell lysates

Cells were recovered by centrifugation, washed twicewith phosphate buffered saline (PBS: 138 mM NaCl,2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4)and resuspended at 2×109 cells ml−1 in 10 mM Tris–Cl pH 7.0 supplemented with protease inhibitors asdescribed [42]. Cells were lysed by three rounds offreeze–thaw and sonication. Extracts were clarified bycentrifugation at 15 000×g for 30 min at 4°C. Low-molecular-mass components were removed by ultrafil-tration using Centricon-3 concentrators (3000 MWcut-off) with three washes with lysis buffer. Proteinconcentrations were determined using the Pierce MicroBCA dye-binding assay.


2.5. Transport assays

Accumulation of [3H]-biopterin and [3H]-folate wasmonitored as described previously [41,43] (Moore et al.,and Moore and Beverley, manuscripts in preparation).Cells were harvested by centrifugation, washed twicewith transport medium (fdM199 medium lacking serumand pteridines [20]), and resuspended at 5×108 cellml−1 in transport medium. Then 100 ml of cell suspen-sion was mixed rapidly with 100 ml of substrate andlayered over a 100 ml cushion of either dibutylphthalate(density 1.043 g ml−1; for promastigotes) or dibutylph-thalate/mineral oil mix (for axenic amastigotes; density1.023 g ml−1). The final concentration of biopterin was200 mM (typical specific activity 10 mCi mmol−1)whereas the final concentration of folate was 10 mM(typical specific activity 150 mCi mmol−1). Triplicatesamples were incubated for various times, at either 23or 4°C, and the reaction terminated by centrifugationthrough the oil layer at 16 000×g for 30 s. Theaqueous layer was aspirated and the oil layer washedtwice with PBS. The cell pellet was recovered, lysedwith 100 ml of Triton X-100 and counted in 5 ml ofScintiverse II scintillation fluid. Net uptake due toactive transport at 23°C was determined by subtractingvalues obtained at 4°C.

2.6. Assay of DHFR-TS and PTR1

Levels of DHFR-TS in cell lysates were measured bythe quantitative binding of [3H]-methotrexate as de-scribed [44,45]. PTR1 activity was determined by twomethods under optimal conditions of 10 mM H2-biopterin as substrate and 100 mM NADPH as cofactorin 20 mM sodium acetate pH 4.7 [20]. First, time-de-pendent oxidation of NADPH was monitored spec-trophotometrically as described [20,21]. Second, anHPLC-based method was developed, which follows H4-biopterin formation by virtue of its differential sensitiv-ity to alkaline treatment relative to the H2-biopterin[46]. Typically, enzyme preparations were incubatedwith H2-biopterin and NADPH in a total volume of500 ml. At intervals (2–20 min) following initiation ofthe reaction, 100 ml aliquots were removed and thereaction terminated by addition of 10 ml of 0.1 M KI/I2,plus either of 5 ml of 1 M HCl or 5 ml of 1 M NaOH.Samples were vortexed briefly and incubated for 1 h at25°C in the dark; then, 10 ml of 1 M HCl was added tothe alkaline samples and the precipitated proteins re-moved by centrifugation at 16 000×g for 2 min. Tothe supernatants, 20 ml of 0.1 M ascorbate was addedfollowed by neopterin as a loading control (2 mM finalconcentration) and the final volume adjusted to 200 mlwith H2O. Samples were separated by HPLC using anoctadecylsilane column with 5% methanol as a mobilephase with a flow rate of 1 ml min−1. Neopterin,

biopterin and pterin were detected by fluorescence usinga Waters 474 Scanning Fluorescence detector, excita-tion and emission wavelengths of 350 and 440 nm,respectively. Products were quantitated against stan-dards whose concentrations were determined from pub-lished extinction coefficients: biopterin, o362

nm=8.3×103 M−1 cm−1 in 0.1 M NaOH; H2-biopterin, o330 nm=6.2×103 M−1 cm−1 at pH 6.8;pterin, o358 nm=7.3×103 M−1 cm−1 in 0.1 M NaOH;neopterin, o362 nm=8.3×103 M−1 cm−1 in 0.1MNaOH [27,46]. Following alkaline treatment, H4-biopterin and H2-biopterin were detected as pterin andbiopterin, respectively whereas following acid treatmentboth H4- and H2-biopterin were oxidized to biopterin[46]. Comparisons of total biopterin obtained after acidtreatment agreed closely with the total pterin plusbiopterin levels obtained after alkaline treatment, indi-cating that the recovery was quantitative.

2.7. Molecular biological methods

Genomic DNA was isolated from late logarithmicphase promastigotes by the LiCl method [47]. Southernblot analysis using 0.7% agarose gels and transfer tonylon membranes was performed as described [48].Blots were hydridized overnight in Church buffer (1%BSA fraction V, 7% SDS, 1mM EDTA, 260 mMsodium phosphate pH 7.2) plus 100 mg ml−1 shearedsalmon sperm DNA at 65°C as described [49]. All blotswere washed twice with 2× SSC (1× SSC is 0.15 Msodium chloride, 15 mM sodium citrate pH 7.0), 0.1%SDS at room temperature for 5 min, twice at 65°C for15 min in the same solution and, finally twice with0.2× SSC, 0.1% SDS at 65°C for 15 min. DNAhybridization probes were made by random-primingusing [a-32P]-dCTP as described [50].

Total RNA was isolated using the phenol/guanidineisothyocyanate reagent TRIzol® (Gibco BRL) accord-ing to the manufacturers instructions. Typically RNAfrom 2×108 cells was extracted using 1 ml of reagent.RNA was separated using a 1% agarose/formaldehydegel and transferred to nylon as described [48]. Blotswere hydridized overnight in Church buffer at 65°C [49]and then washed twice with 2× SSC, 0.1% SDS atroom temperature for 5 min followed by two washes at65°C for 15 min in the same solution. PTR1 andDHFR-TS-probed blots were subsequently washedtwice with 0.2× SSC, 0.1% SDS at 65°C for 15 min.Relative intensities of hybridizing transcripts were de-termined by densitometry.

2.8. Cloning of the L. major Friedlin V1 BT1 gene

A L. major Friedlin V1 cosmid library made in thevector cLHYG [51] was probed with the L. dono6aniBT1 gene and six overlapping cosmids were isolated.


From two BT1-containing cosmids (5.1.1 and 6.1.1,lab. strain numbers B4032 and B4033, respectively) theL. major BT1 gene was identified and sequenced.

3. Results

3.1. An HPLC-based assay for measurement of PTR1acti6ity in cell lysates

PTR1 but not DHFR-TS is able to reduce H2-biopterin, potentially allowing the measurement ofPTR1 and DHFR separately in crude parasite extracts[20]. However, the low levels of PTR1 activity in totalcell extracts rendered the standard NADPH-based spec-trophotometric method inadequate [20,21]. We devel-oped a sensitive alternative assay, where thetime-dependent formation of H4-biopterin was moni-tored by HPLC. Following enzymatic synthesis, theproduct H4-biopterin is converted to pterin under con-ditions of alkaline oxidation, whereas the substrateH2-biopterin is oxidized to biopterin (Fig. 2A; Ref.[46]). These two forms are readily separated by HPLCand quantitated by fluorescence (Fig. 2B).

The HPLC assay was validated with purified L.major PTR1 [20]. With H2-biopterin as substrate,purified PTR1 showed a time-dependent increase inpterin formation (derived from the product H4-biopterin) and concomitant decrease in the amount ofbiopterin (derived from the substrate H2-biopterin; Fig.2B and C). Pterin formation was not observed follow-ing alkaline treatment if PTR1 was omitted (data notshown). Acid treatment of the samples, which yieldsbiopterin from both oxidized and reduced biopterins,showed that the total biopterin levels were quantita-tively equal to the sum of the pterin and biopterinunder alkaline conditions (data not shown). Thus, theHPLC based method provided a faithful readout ofH4-biopterin levels. Significantly, the rates of pterin(H4-biopterin) formation and biopterin (H2-biopterin)loss were equivalent, as expected, and linear with time

Fig. 2. (Continued)

Fig. 2. An HPLC method for measurement of PTR1 activity. (A)Scheme for the differential alkaline oxidation of H2-biopterin (H2B)and H4-biopterin (H4B), yielding biopterin or pterin respectively. (B)PTR1-dependent formation of pterin. Aliquots were removed from aPTR1 assay every 5 min, subjected to alkaline oxidation and analyzedby HPLC as described in Section 2. Bona fide standards were used toidentify and quantitate the pteridines. (C) Time course of PTR1activity. The decrease in substrate H2-biopterin (detected as biopterin;) and the concomitant increase in the product H4-biopterin (de-tected as pterin; �) at various times following addition of PTR1 isshown. (D) Comparison of PTR1 activities measured by either theHPLC (x-axis) or spectrophotometric (y-axis) methods. In partsB–D, assays contained 100 mM NADPH, 10 mM H2-biopterin, 20mM sodium acetate pH 4.7, and purified L. major PTR1 (0.5 mg inparts B and C; 0.05–1 mg in part D), in a total volume of 500 ml.


(Fig. 2C) and added PTR1 (up to saturating values;data not shown).

These data suggested that the HPLC method wassuitable for quantifying PTR1 activity. We comparedthe results of the HPLC assay with that of the spec-trophotometric assay, using serial dilutions of purifiedL. major PTR1 (Fig. 2D). The two assays were in goodagreement over the range of product formation of 1–40pmol min−1. Outside of this range the activities deter-mined using the HPLC method deviates significantlyfrom linearity (not shown). At the low end, theamounts of pterin formed approach the limits of detec-tion and spontaneous oxidation of the H4-biopterinbecomes proportionately significant. At the high end,substrate depletion becomes a factor; in these circum-stances, product formation can be measured over ashorter period of time.

We tested the performance of the HPLC assay withLeishmania crude extracts. Previously we reported thecreation of null mutant parasites lacking PTR1 [20,21].As expected, cell extracts (5 mg protein ml−1) from theL. major ptr1− parasites showed no measurable H2-biopterin reductase activity in the HPLC assay (notshown). We then added purified PTR1 in increasingconcentrations to the ptr1- extract; H2-biopterin reduc-tase activity was observed and was quantitatively iden-tical to that obtained with purified PTR1 aloneindicating that the assay was unaffected by the presenceof the cell extract (data not shown).

In previous studies, the spectrophotometric assay ofPTR1 activity in crude lysates of wild-type, ptr1− andPTR1-overexpressing parasites yielded relative activi-ties of 1 : 0.2 : 3.2 [21], while western blot analysis withanti-PTR1 antisera suggested a relative abundance of1:0:100 [20]. In contrast, with the HPLC assay wedetermined PTR1 activity with these three lines to be21, B2 (below the limit of detection) and 1480 pmolmin−1 mg−1 soluble protein, or 1 : B0.1 : 70 (wild-type:ptr1−:PTR1-overexpressor), in good agreementwith the western blot values. Thus, the HPLC-basedassay provides a sensitive and robust method for deter-mining PTR1 activities in crude Leishmania extracts,without interference from other activities. It is now thepreferred method and was employed in all followingstudies.

3.2. Growth-phase and de6elopmental regulation ofPTR1

In promastigotes from all species, the highest PTR1activity was seen in logarithmically growing cells (10–180 pmol min−1 mg−1 soluble protein; Fig. 3A). Thelevels decreased somewhat in stationary phase cells, toapproximately 70% of log phase values (Fig. 3A). Simi-lar changes in protein levels were seen in western blotstudies with L. dono6ani and L. major (data not shown;

Fig. 3. Expression of PTR1 activity and mRNA during growth anddevelopment. (A) PTR1 activity was measured using the HPLCmethod. For each species and growth stage (indicated at the bottomof panel B), at least two independent cell extracts were examined,with multiple points falling within the linear range of the assay. Themeans and standard deviations are shown. (B) Northern blot analysisof RNAs from different stages and/or species of Leishmania. Fivemicrograms of total RNA from each sample was analyzed andhybridized with a L. major PTR1 full-length coding region probe; theethidium bromide stained gel showing the three rRNAs (the largesubunit rRNA of Leishmania known to be cleaved into two largefragments) is provided as a loading control.

studies with L. mexicana were not feasible due to poorcross-reactivity of the anti-L. major PTR1 antibodywith L. mexicana PTR1). We were able to obtainsufficient quantities of purified infective L. major meta-cyclic promastigote to permit PTR1 activity measure-ments, which showed values similar to total stationaryphase cells (7.2 and 8.6 pmol min−1 mg−1). Promastig-ote levels of PTR1 in L. major were 10 to 17-fold lowerthan observed in L. dono6ani and L. mexicana,respectively.

Analysis of cultured L. mexicana amastigotes demon-strated for the first time the presence of PTR1 activityin this stage of the parasite, at levels about fourfoldlower than in promastigotes. Unlike promastigotes,amastigote PTR1 levels did not decline in stationaryphase (Fig. 3A).


3.3. Growth-phase and de6elopmental regulation ofDHFR-TS

The abundance of DHFR-TS in crude cell lysateswas determined by stoichiometric binding of [3H]-methotrexate [41,44,45]. All species contained similarlevels of DHFR-TS in logarithmic phase promastigotes,ranging between 10 and 16 pmol mg−1 soluble protein.Levels in stationary phase were only slightly less thanfound in log phase (Fig. 4A). L. mexicana amastigotesexpressed tenfold less DHFR-TS than promastigotes,and in both stages there was no significant difference inDHFR-TS levels between cells in the logarithmic stageand after 48 h in stationary phase. Overall these resultsare consistent with previous studies of DHFR-TS inLeishmania [4,20,45,52].

Fig. 5. Kinetics of biopterin and folate accumulation by L. mexicana.The net accumulation of (A) biopterin or (B) folate of logarithmicpromastigotes (�) or logarithmic amastigotes () is shown (meansand S.D.s of three samples each). External concentrations ofbiopterin and folate were 200 and 10 mM, respectively.

Fig. 4. Expression of DHFR-TS and mRNAs in growth and develop-ment. (A) The abundance of DHFR-TS was determined by quantita-tive binding of [3H]-methotrexate as described in the methods. Valuesrepresent the average of two independent extracts each measured induplicate. (B) Northern blot analysis of total RNA hybridized withthe L. major DHFR-TS full-length coding region probe, as describedin the legend to Fig. 3B. ND, not done.

3.4. Kinetics of biopterin and folate uptake

The time-dependence of biopterin accumulation wasexamined in L. mexicana, L. dono6ani and L. major.Typical profiles for log-phase L. mexicana promastig-otes and amastigotes are shown in Fig. 5A (similar dataobtained with L. dono6ani and L. major promastigotesare not shown). Promastigotes rapidly accumulated[3H]-biopterin, with linear kinetics over the first 5 minand reaching a plateau value after 60 min (Fig. 5A,open circles), as seen previously [3,9]. A different profilewas observed with log-phase amastigotes, with an ini-tial linear phase for the first 10 min, followed by asteady decrease to about 25% of the peak values (Fig.5A, closed circles).

The time-dependence of folate accumulation was ex-amined in L. mexicana, L. dono6ani and L. major. Atypical profile for log-phase L. mexicana axenicamastigotes and promastigotes is shown in Fig. 5B


(similar data obtained with L. dono6ani and L. majorpromastigotes are not shown). As seen with biopterin,[3H]-folate accumulation was rapid and accumulatedwith linear kinetics for the first 5 min, until a maximalconcentration was attained. Subsequently, intracellular[3H]-folate decreased until after 60 min, only 30 and 8%of the maximal levels remains for promastigotes andamastigotes, respectively.

Thus while promastigotes show simple uptake kinet-ics for biopterin, amastigotes with biopterin and bothstages with folate exhibit more complicated profiles.Similar results were reported previously in murineleukemia cell L1210 [53] and L. major [41]. In murinecells this profile was attributed to the presence ofradiolabeled breakdown products of folate, however, inL. major this was ruled out by carefully purifying allradiolabeled ligands [41]; similar procedures were fol-lowed here (Section 2). The source of this phenomenonis not known, and probably reflects some aspect ofpteridine metabolism as yet uncharacterized [41]. As inprevious studies, we used the initial linear uptake phaseas a measure of transporter activity (Figs 6A, 7A).Previous studies of mutant promastigotes showed thatall biopterin uptake activity can be attributed to thetransporter encoded by BT1 [8,9] (Cunningham andBeverley, unpublished data), while 99% of folate uptakein L. dono6ani can be attributed to the transporterencoded by FT1 (Moore and Beverley, and Moore etal., manuscripts in preparation).

3.5. Growth phase and de6elopmental regulation ofpteridine accumulation

Log-phase promastigotes of all three species dis-played similar rates of biopterin uptake, ranging be-tween 94 and 210 pmol−1 min−1 109 cells−1 (Fig. 6A).These values agree well with previous studies of L.dono6ani and L. major [3,8] (Moore et al., manuscript inpreparation) and L. tarentolae (data not shown). Ascells entered stationary phase biopterin uptake declined,to 16–27% of log-phase values after 48 h in stationaryphase (Fig. 6A). Cultured L. mexicana amastigotesshowed 30% higher rates of biopterin uptake thanpromastigotes. As with promastigotes, biopterin trans-port activity decreased in stationary phase amastigotes,albeit to a lesser extent (67% maximal; Fig. 6A).

Similar results were obtained with folate uptake in allthree species. Log-phase promastigotes showed thehighest rate of uptake, ranging from 6.2 to 23 pmol−1

min−1 109 cells−1. L. major and L. dono6ani exhibitedrates of folate uptake about threefold higher than L.mexicana (Fig. 7A). As parasites entered stationaryphase, the rate of folate uptake declined, to 10–22% oflog-phase values (Fig. 7A). Log-phase L. mexicanaamastigotes accumulated folate with rates comparableto that of promastigotes (5.4 pmol−1 min−1 109

cells−1; Fig. 7A), and as with biopterin, this rate de-clined by 30% in stationary phase amastigotes.

3.6. Regulation of BT1 RNA expression

The L. major Friedlin V1 BT1 gene was cloned andsequenced. An open reading frame of 1893 bp wasidentified, predicting a BT1 protein of 631 residueswhich showed 9.3% and 11.8% amino acid differencewith L. dono6ani and L. mexicana BT1, respectively[8–10]. Southern blot data showed that this gene wassingle copy in L. major and corresponded to the BT1

Fig. 6. Biopterin uptake and BT1 RNA during growth and develop-ment. (A) The initial rate of biopterin uptake was measured asdescribed in the methods. Each determination represents the averageof triplicate determinations of three different experiments; the meansand standard deviations are shown. The external biopterin concentra-tion was 200 mM. ND, not done. (B) Northern blot analysis of totalRNA hybridized with the L. major BT1 full-length coding regionprobe, as described in the legend to Fig. 3B. RNA standards areshown to the left and BT1 transcript sizes to the right.


Fig. 7. Folate uptake and expression of FT1-related RNAs during growth and development. (A) The initial rate of folate uptake was measuredas described in the methods. Each determination represents the average of triplicate determinations of three different experiments; the means andstandard are deviations shown. The external folate concentration was 10 mM. ND, not done. (B) Northern blot analysis of total RNA hybridizedwith the L. dono6ani DI700 FT1 full-length coding region probe, as described in the legend to Fig. 3B. RNA standards are shown to the left andtranscript sizes to the right.

gene recently found on chromosome 35 (Fig. 8A; [75] inpress). Similarly, a single copy of BT1 was found in thestrains of L. mexicana and L. dono6ani studied here, asdescribed in other strains of these species (Fig. 8A;Refs. [8,9] (Moore et al., manuscript in preparation).

Northern blot analysis showed that while only asingle BT1 mRNA was evident in L. major, there weretwo in L. dono6ani and four in L. mexicana (Fig. 6B).Given that BT1 is a single copy gene in all species, themultiple transcripts must be precursors or arise from


alternative RNA processing. Notably, in L. mexicanathe 3.5 kb RNA was expressed in both stages, the 4.7and 2.4 kb RNAs were predominately expressed inpromastigotes, and the 2.9 kb RNA was restricted toamastigotes (Fig. 6B).

In L. major and L. mexicana promastigotes, theoverall levels of BT1 RNA(s) followed biopterin (BT1)uptake activity, with log phase showing higher levelsthan stationary phase promastigotes (Fig. 6A, B). Incontrast, L. dono6ani BT1 RNA showed little stagevariation (Fig. 6B).

3.7. Regulation of FT1 family RNA expression

In L. dono6ani, there are at least six genes related toFT1 present in a single genomic locus, although only

FT1 has been associated with folate transporter activity(Moore and Beverley, manuscript in preparation).While the structure of the FT1 locus was not deter-mined for L. major and L. mexicana, Southern blotanalysis with six different enzymes suggested that multi-ple genes are present in these species as well (Fig. 8B).Since gene-specific probes were not available, we usedan L. dono6ani FT1 probe to assess the expression oftranscripts arising from the FT1 family.

The expression profiles of FT1-related RNAs wascomplex, with three to seven prominent transcriptsevident in each species (Fig. 7B). These RNAs may beprecursors or arise from alternative RNA processing,however we favor the possibility that they arise fromdifferent FT gene family members. Despite this com-plexity, several patterns of RNA expression were evi-dent. Within promastigotes, most FT1-related RNAsdecreased in abundance after 48 h in stationary phaserelative to logarithmic phase (Fig. 7B). However, therewere exceptions: in L. major an 8.5 kb RNA increasedin stationary phase promastigotes while a 3.5 kb RNAwas uniquely detected in metacyclic promastigotes (Fig.7B). Several RNAs showed little or no changes inabundance between log and stationary phase L. dono-6ani (2.7, 3.3 and 3.6 kb; Fig. 7B).

Comparisons of L. mexicana promastigotes andamastigotes showed that most of the FT1-related tran-scripts detected in promastigotes were shared byamastigotes as well. However, three transcripts (7.0, 4.3and 3.9 kb) were up-regulated, in some cases nearlytenfold more abundant than in log-phase promastig-otes. There was little or no change in FT1-related RNAabundance between logarithmic and stationary phaseamastigotes (Fig. 7B). Thus, FT1-related RNA expres-sion is complex, and likely to involve differential ex-pression and/or processing of individual gene familymembers.

3.8. Regulation of PTR1 and DHFR-TS mRNAexpression

PTR1 is a single copy gene and a single mRNA of1.5–1.6 kb was detected for all species examined (Fig.3B) [24,25]. In contrast to PTR1 activity, the abun-dance of PTR1 mRNA decreased eight to 13-fold after48 h in stationary phase in all three species (Fig. 3B),although purified metacyclic stage L. major showedPTR1 mRNA levels closer to log-phase promastigotes(Fig. 3B). Lastly, PTR1 mRNAs in logarithmic andstationary phase L. mexicana amastigotes were equallyabundant, and similar to the levels in logarithmic pro-mastigotes (Fig. 3B).

DHFR-TS is a single copy gene, and a single 3.1–3.3kb mRNA was identified in Northern blot analysis ofall species (Fig. 4B) [14]. Maximal mRNA levels weredetected in logarithmic promastigotes, which decreased

Fig. 8. Southern blot analysis of L. major, L. dono6ani and L.mexicana genomic DNA. Genomic DNA from the species indicatedwas digested with restriction enzymes and subjected to Southern blothybridization. The enzymes used were: lane 1, EcoRI; lane 2,HindIII; lane 3, NheI; lane 4, NotI; lane 5, SacI; and lane 6, SpeI.(A) L. major V1 BT1 full-length coding region probe. (B) L. dono6aniDI700 FT1 full-length coding region probe.


between six and 20-fold after 48 h in stationary phase.Expression of DHFR-TS mRNA in amastigotes resem-bled promastigotes, with maximal levels being presentin logarithmic growing cells and decreasing sixfold after48 h in stationary phase. Thus, as with PTR1, thedecrease in mRNA during promastigotes growth is notreflected by equally large decreases in protein abun-dance. Furthermore, whilst the mRNA levels in loga-rithmic amastigotes is approximately 60% of that seenin logarithmic promastigotes, the abundance of proteinis tenfold lower.

4. Discussion

Nutritional, biochemical and genetic studies showthat both conjugated (folates) and unconjugated(biopterin) pteridines are essential for Leishmaniagrowth [3–7,17,20,21,54]. Fortunately for the parasite,both its insect and mammalian hosts have well-devel-oped pathways for the synthesis of unconjugatedpterins de novo [27] and/or for the recovery and utiliza-tion of folates from the diet [11,12]. Thus, throughoutthe infectious cycle Leishmania has only to solve thetask of exploiting the pteridine resources availablewithin its hosts. Correspondingly, our data show thatLeishmania expresses all known activities required forpteridine salvage and utilization throughout its infec-tious cycle.

4.1. Pteridine uptake is regulated by growth phase butnot de6elopment

The first step in pteridine salvage is uptake (Fig. 1).Our data show that both biopterin and folate uptakeoccurs at high levels in the amastigote stage of L.mexicana, comparable to that observed in the pro-mastigote stage (Figs. 6 and 7) [3,8,9,41,55] (Moore etal., and Moore and Beverley, manuscripts in prepara-tion). Since amastigotes normally reside within a para-sitophorous vacuole within vertebrate macrophages, weinfer that both transporters must act within this cellularcompartment during a natural infection. BT1 is theonly biopterin transporter known in Leishmania (or anyother organism currently), and is likely to be responsi-ble for biopterin uptake in vivo. In contrast, Leishma-nia possess a family of transporters related to FT1(Moore and Beverley, manuscript in preparation), andactivity and Northern blot data suggest that one ormore members of this family are acting in the amastig-ote and metacyclic stages (Fig. 7B). In other Leishmaniagene families, diversity in the structure, activity andregulation of individual members has been observed(such as the glucose transporters and the surfaceprotease gp63 [56,57]). Resolution of this question forthe FT1 gene family awaits the development and appli-

cation of gene-specific probes, and/or the genetic dis-ruption of the other FT1-related genes and tests withinthe amastigote stage.

Folate and biopterin transport activities are similarin promastigotes and amastigotes (Figs 6A, 7A). Incontrast, these activities decline substantially as thedividing parasites enter stationary phase (Figs 6A, 7A)[41]. This effect is strongest in the promastigote stage,where on average an 80% and 85% decrease in BT1 andFT1 activity was observed, respectively. Thus it is evi-dent that Leishmania is able to differentially regulatepteridine uptake in response to growth phase and devel-opmental signals. Notably, cessation of growth inLeishmania acts as a trigger for differentiation into theinfective metacyclic promastigote phase of the parasite[58]. Although the role of biopterin in parasite growthis unknown, down-regulation of folate transporter ac-tivity may be associated with the formation of folatepolyglutamates which are efficiently retained within thecell [59], and/or the cessation of growth and DNAsynthesis.

4.2. Alterations in pteridine uptake associated withadaptation to in 6itro culture

While the rate of folate accumulation determinedhere with L. dono6ani agrees well with previous studies[3], the rate of folate uptake observed in the highlyvirulent L. major strain Friedlin V1 was more thantenfold less than observed previously with anotherstrain, CC1 (23 versus 270 pmol−1 min−1 109 cells−1

[41]). We repeated the studies of L. major CC-1 andconfirmed the original finding (data not shown). Previ-ous studies have noted differences in antifolate sensitiv-ity and folate pathways between the fully virulentstrains of L. major (Friedlin V1 and LV39) and attenu-ated derivatives thereof [60,61], and L. major strainCC-1 is itself somewhat less virulent in mouse infections[18]. Similarly, changes in biopterin uptake and BT1expression have been associated with adaptation to invitro culture, due to amplification and/or rearrange-ments of the BT1 gene in L. dono6ani [8,10] and L.major (Moore et al., manuscript in preparation), aprocess distinct from alterations seen in methotrexate-selected L. tarentolae [9]. These findings underscore theimportance that the Leishmania parasite places on themaintenance of sufficient intracellular pteridine levels.Moreover, it establishes the need to focus on virulentstrains when investigating the role of pteridinemetabolism in the natural infectious cycle.

4.3. PTR1 and DHFR-TS are expressed throughoutthe infectious life cycle

Following entry into the cell, biopterin and folatemust be reduced to the active H4-forms. Biopterin


reduction is carried out exclusively by PTR1, whilefolate reduction in promastigotes is mediated predomi-nantly by DHFR-TS and to a lesser extent by PTR1(Fig. 1) [20,22,62]. We found it necessary to develop amore sensitive assay for PTR1 activity that would befree from interference by contaminating activitiespresent in parasite crude extracts. An HPLC-basedmethod was developed, validated, and shown to besuperior to the spectrophotometric method used previ-ously by several criteria, including sensitivity and free-dom from interference in crude lysates.

During development, DHFR-TS levels in L. mexi-cana amastigotes declined considerably relative to pro-mastigotes, decreasing tenfold (Fig. 4A), comparable tothe sixfold decline reported previously in lesion L.major amastigotes [45]. In contrast, PTR1 levels de-clined only threefold in L. mexicana amastigotes rela-tive to logarithmic promastigotes (Fig. 3A). Thepotential implications for chemotherapy are discussedlater. In contrast to L. mexicana, amastigotes of therelated trypanosomatid parasite Trypanosoma cruzi donot express PTR1 [63]. This may be related to fact thatT. cruzi amastigotes reside within the cytoplasm andthus have free access to reduced biopterin in the cyto-plasm, unlike Leishmania amastigotes which residewithin a membrane-enclosed vacuole.

Neither PTR1 nor DHFR-TS showed substantialreductions in expression related to growth phase, inpromastigote or amastigotes (Figs 3A, 4A). Overall,our findings indicate that pteridine uptake activitiesappear to be regulated more strongly by growth phasethan development, while pteridine reductase levels aremore strongly affected by development than growthphase.

4.4. Post-transcriptional mechanisms play a major rolein regulation of pteridine sal6age acti6ities

As the first step towards understanding the geneticregulatory mechanisms controlling pteridine salvage, weexamined RNA levels during the growth cycle anddevelopment of Leishmania. In some cases, a reason-able correlation between changes in activity and RNAlevel were observed (BT1 in L. major and L. mexicana ;possibly FT1 in L. mexicana). More commonly andsomewhat unexpectedly, RNA and activity levels werenot strongly correlated. In some cases, activity re-mained high while RNA levels declined (stationaryphase promastigote PTR1 and DHFR-TS levels in allspecies), while in others activity declined while RNAlevels remained high (FT in L. major and L. dono6ani ).Remarkably, the pattern of RNA/activity regulationshowed variation even between species, often while thepattern of activity was conserved (BT1 in L. dono6ani,relative to L. major and L. mexicana).

Leishmania, like other trypanosomatid parasites, em-ploy a polycistronic transcriptional mechanism in whichlarge precursors are processed by coupled 5% trans-splic-ing and 3% polyadenylation to form mature mono-cistronic RNAs [64–66]. Consequently,post-transcriptional mechanisms of gene regulationmust play a larger role in general [66], and perhaps thisis responsible for the diversity of regulatory patternsseen in the pteridine salvage pathway of Leishmania.Several studies have stressed the importance of bothtranslational regulation and protein turn-over (for ex-ample, Refs. [67,68]), and these could respectively ac-count for the ‘high RNA/low protein’ and ‘highprotein/low RNA’ patterns seen in pteridine salvageregulatory patterns noted above. While post-transla-tional modification of proteins may also play a role,preliminary data with anti-PTR1 antisera suggestedthat protein and activity levels were in good agreement.Interestingly, the monofunctional DHFR and TS frommammals have been shown to bind their respectivemRNAs and inhibit translation, suggesting another po-tential mode of regulation [69,70].

In total, these data provide evidence for an unex-pected complexity in the mechanisms employed to regu-late pteridine salvage at both the RNA and proteinlevels, amongst genes and amongst different pathogenicLeishmania. These pathways will provide a rich groundsfor future studies of regulatory mechanisms and diver-sity amongst parasite species that seemingly live withinsimilar evolutionary niches. Lastly, it suggests that theunderstanding of the role of these pathways in parasitemetabolism will depend heavily upon direct measure-ment of relevant enzymatic/transporter activities, ratherthan RNA levels.

4.5. Implications for chemotherapy

In previous studies of the promastigote stage of L.major, we hypothesized that PTR1 expression couldprovide a potential ‘metabolic by-pass’ of DHFR-TSinhibition, allowing a partial or complete reversal ofanti-pteridine inhibition depending upon the relativelevel of PTR1 expression [20,21]. This is due primarilyto the insensitivity of PTR1 to anti-pteridines relativeto DHFR, permitting folates to be reduced even underconditions of DHFR inhibition [20,21,71]. Our dataextend the PTR1 by-pass model to other pathogenicstrains and also to the chemotherapeutically relevantamastigote stage. Firstly, the PTR1 activity in L. majorpromastigotes is 10 to 17-fold lower than in L. dono6aniand L. mexicana, and L. major is considerably moresensitive to methotrexate. This suggests that the role ofPTR1 as a metabolic-bypass is likely to be even greaterin L. dono6ani and L. mexicana than L.major [6,72].Secondly, the data presented here also show that cul-tured L. mexicana amastigotes express high levels of


PTR1, much higher than observed previously in L.major promastigotes (Fig. 3A), again providing ampleevidence for the presence of the PTR1 ‘by-pass’ path-way in amastigotes. Moreover, DHFR-TS levels arelower in L. mexicana amastigotes relative to promastig-otes (Fig. 4A), suggesting that if anything the PTR1-de-pendent effects will be exacerbated relative to that seenin promastigotes. No PTR1-like activity has been de-tected in the other protozoa for which anti-folate basedchemotherapy has proven successful.

Thus, in order to be effective, anti-folate chemother-apy targeting the macrophage-resident amastigote stageof Leishmania will need to take into account the abilityof PTR1 to by-pass inhibitors targeted against DHFR-TS. One possibility is the identification of compoundsable to inhibit both PTR1 and DHFR-TS simulta-neously, or separately. Preliminary studies have iden-tified several lead compounds which inhibit both PTR1and DHFR enzymes in vitro, and inhibit promastigotegrowth at sub-micromolar concentrations [71], and onecompound inhibited the growth of L. major amastigoteswithin cultured macrophages (Nare B, Beverley SM,unpublished observation). The three-dimensional struc-tures of both L. major DHFR-TS and PTR1 have nowbeen solved, and in combination with the structure ofthe human DHFR, will facilitate the design of newinhibitors selective for parasite but not host enzymes[73,74]. Another possibility involves blocking folateand/or biopterin uptake, through inhibition of the BT1and/or FT1 transporters, perhaps in conjunction withanti-pteridine reductase inhibitors.

In summary, our studies present a comprehensivepicture of the pteridine salvage pathway, across thegrowth and developmental stages of Leishmania. Thedevelopmental changes observed pose intriguing ques-tions about the roles of pteridines in the complex lifecycle of this parasite and the regulatory mechanismsunderlying their control. Furthermore, the demonstra-tion that amastigotes possess an extensive pteridinesalvage pathway comparable to that of promastigoteshas profound implications for anti-folate basedchemotherapy, and may explain the failure to success-fully exploit this essential pathway.

Acknowledgements

We thank D. Dobson, T. Ellenberger, F. Gueiros-Filho, L.-F. Lye, J. Moore, B. Nare, K. Zhang, and D.Zilberstein for advice, discussions, permission to men-tion unpublished data, and comments on thismanuscript. Supported by NIH grants AI21903 andAI29646.

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Pteridine salvage throughout the Leishmania infectious cycle

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